Among physiologically active substances or environmental pollutants such as natural products, toxins, hormones or agricultural chemicals, numerous substances act in ultratrace amounts. Accordingly, instrumental analytical methods capable of performing highly sensitive analysis have conventionally been widely used for qualitative and quantitative measurement of these substances. However, instrumental analytical methods are poor in specificity, require excessive time for analysis including pretreatment of samples, and are troublesome in operation. Thus, instrumental analytical methods are inconvenient for the purpose of rapid and convenient measurements that have been required in recent years. Meanwhile, immunoassays are highly specific and much easier in terms of operation than instrumental analytical methods. Therefore immunoassays have gradually spread in the field of measurement of bioactive substances and environmental pollutants. However, conventional immunoassays such as enzyme immunoassays using 96-well plates and latex agglutination assays do not always provide satisfactory rapidness and convenience for measurement or detection sensitivity.
Another need expected to be enabled is as follows. Achievement of higher sensitivity of tests that currently use relatively invasive samples such as swabs and blood makes it possible to detect very small amounts of analytes contained in relatively low-invasive samples such as snot, mouth wash, and urine. Thus, less burdensome tests of patients can be realized.
In recent years, test kits using an immunochromatography method (hereinafter referred to as an immunochromatographic kit) have been used more often in examination of infections that require particularly rapid diagnosis. According to the spread of these kits, patients with infections can be identified by a rapid and convenient method, and subsequent diagnosis and therapy can be conducted immediately and accurately. For example, in an immunochromatography method using the sandwich method, a labeled second antibody (second binding substance) capable of specifically binding to an analyte (for example, an antigen), and a sample solution which may possibly contain the analyte are developed on an insoluble thin film-shaped support (for example, a glass fiber membrane, a nylon membrane, or a cellulose membrane) on which a first antibody (first binding substance) capable of specifically binding to the analyte has been immobilized in a specific region. As a result, an immune complex with the analyte is formed at the region of the insoluble thin film-shaped carrier, on which region the first antibody has been immobilized. The analyte can be measured by detecting a signal such as color development or coloring of a label. The label to be used herein may be, for example, a protein such as an enzyme, colored latex particles, metal colloids, or carbon particles.
The immunochromatography method requires neither massive facilities nor instruments for determination and measurement. Furthermore, the immunochromatography method is simple in operation and promptly gives measurement results by introducing a sample solution dropwise which may possibly contain an analyte and leaving it for approximately 5 to 10 minutes. For this reason, this technique is used widely as a convenient, rapid, and highly specific method for determination and measurement in many scenarios, such as for clinical examination in hospitals and in assays in laboratories.
Among physiologically active substances or environmental pollutants such as natural products, toxins, hormones and agricultural chemicals, many substances exert effects in ultratrace amounts that are undetectable by conventional common immunochromatography methods. Therefore, there are demands for development of rapid, convenient, and highly sensitive immunochromatography methods for such substances.
Known examples of an immunochromatography method involving signal amplification and highly sensitive assay include an enzymatic amplification method (JP Patent No. 3309977) and a silver amplification method (JP Patent Publication (Kokai) No. 2002-202307 A). There have been commercially available products for immunochromatography methods involving enzymatic amplification. JP Patent No. 3309977 describes amplification after washing. In such case, highly sensitive assay can be achieved to a greater extent than that in a conventional immunochromatography method using a metal label or colored latex particles. However, in the above case, enzymatic amplification requires time for reaction and thus measurement is time-consuming compared with the conventional cases that use labels, which is disadvantageous.
In the case of immunochromatography involving silver amplification, the background level derived from metal label particles must be reduced in order to obtain a favorable signal/noise (S/N) ratio. Therefore, it is necessary to carry out washing in order to increase the final detection sensitivity. In a system to which a membrane for washing is previously provided, an antigen solution accidentally flows in the direction of a second insoluble carrier or a third insoluble carrier upon development of the antigen solution, resulting in loss of labeled antibodies, leading to a decrease in sensitivity. In addition, labels that have accidentally flowed in the direction of a second insoluble carrier cause an increase in the background level. Eventually, the S/N ratio derived from the detection level and the background level decreases and thus the detection sensitivity decreases, which is problematic.